A deficient DNA mismatch repair (MMR) system is observed in about 10-15% of all colorectal carcinomas and in up to 90% of hereditary non-polyposis colorectal cancer (HNPCC) patients. Tumors with MMR defects acquire mutations in short repetitive DNA stretches, a phenomenon termed microsatellite instability. The determination of microsatellite status in colon cancer is of increasing relevance, since (1) microsatellite status is an independent prognostic factor in colorectal cancer, (2) the efficacy of adjuvant chemotherapy seems to be dependent on microsatellite status of the tumor, and (3) microsatellite instability is the most important molecular screening tool for the identification of HNPCC patients and families affected by germline mutations in MMR genes. Therefore, routine MSI testing appears to be justified for all colorectal cancer cases.
Microsatellite instability is observed in about 10-15% of sporadic colorectal carcinomas (CRCS) and in up to 90% of hereditary non-polyposis colorectal cancer (HNPCC) patients that harbor germline mutations in DNA mismatch repair (MMR) genes (for a review see Lynch and de la Chapelle “Hereditary colorectal cancer”, N Engl J Med. 2003 Mar. 6; 348(10):919-932). CRCs displaying the microsatellite instability (MSI) phenotype possess particular pathological and clinical features. MSI-H CRCs are often localized in the proximal colon and present with a dense intratumoral lymphocyte infiltration (Smyrk et al. “Tumor-infiltrating lymphocytes are a marker for microsatellite instability in colorectal carcinoma, Cancer 2001 Jun. 15; 91(12):2417-22; Dolcetti et al. “High prevalence of activated intraepithelial cytotoxic T lymphocytes and increased neoplastic cell apoptosis in colorectal carcinomas with microsatellite instability, Am J Pathol. 1999 June; 154(6):1805-13). Several studies report a better prognosis for MSI-H CRC patients (Gryfe et al. “Tumor microsatellite instability and clinical outcome in young patients with colorectal cancer”, N Engl J Med. 2000 Jan. 13; 342(2):69-77; Wright et al. “Prognostic significance of extensive microsatellite instability in sporadic clinicopathological stage C colorectal cancer”, Br J Surg. 2000 September; 87(9):1197-202); Samowitz et al. “Microsatellite instability in sporadic colon cancer is associated with an improved prognosis at the population level”, Cancer Epidemiol Biomarkers Prev. 2001 September; 10(9):912-23). Interestingly, the susceptibility towards chemotherapy seems to be dependent on microsatellite status of colorectal tumor cells (Claij, “Microsatellite instability in human cancer: a prognostic marker for chemotherapy?”, Exp Cell Res. 1999 Jan. 10; 246(1):1-10); Hemminki et al. “Microsatellite instability is a favorable prognostic indicator in patients with colorectal cancer receiving chemotherapy, Gastroenterology. 2000 October; 119(4):921-8; Watanabe et al. “A change in microsatellite instability caused by cisplatin-based chemotherapy of ovarian cancer”, Br J Cancer 2001 Sep. 28; 85(7):1064-9). The DNA MMR system appears to be involved in apoptosis induction via DNA-damaging agents, in vitro, several cell lines with a defective mismatch repair system have been shown to be resistant to such agents (Claij “Microsatellite instability in human cancer: a prognostic marker for chemotherapy?”, Exp Cell Res. 1999 Jan. 10; 246(1):1-10; Bawa and Xiao “A mutation in the MSH5 gene results in alkylation tolerance, Cancer Res. 1997 Jul. 1; 57(13):2715-20”; Carethers et al. “Mismatch repair proficiency and in vitro response to 5-fluorouracil, Gastroenterology 1999 July, 117(1):123-31”). In a study of Ribic et al. (“Tumor microsatellite-instability status as a predictor of benefit from fluorouracil-based adjuvant chemotherapy for colon cancer”, N Engl J Med. 2003 Jul. 17; 349(3):247-57), a tendency towards a shorter overall survival was observed in 5-fluorouracil (5-FU) chemotherapy-treated patients with MSI-H CRC, whereas patients with MSS CRC benefited from adjuvant 5-FU therapy. In a different study, the improved survival of CRC patients treated with chemotherapy was restricted to MSS cases, whereas no effect was detected in the MSI-H group (Carethers et al. “Use of 5-fluorouracil and survival in patients with microsatellite-unstable colorectal cancer”, Gastroenterology 2004 February; 126(2):688-9).
These data point to the clinical significance of microsatellite status in CRC and provide good reasons for routine MSI testing of all colorectal cancer cases. The current standard method however is time-consuming, laborious, and expensive.
At present, MSI-testing is usually only applied to patients preselected upon clinical criteria (Bethesda guidelines, Boland et al. “A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer”, Cancer Res. 1998 Nov. 15; 58(22):5248-57), because the standard testing procedure recommended by the ICG-HNPCC workshop (Boland et al. “A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer”, Cancer Res. 1998 Nov. 15; 58(22):5248-57),) implies a considerable laboratory workload: Five microsatellite markers including two mononucleotide repeats (BAT26 and BAT25) and three dinucleotide repeats (D2S123, D5S346, D17S250) have to be amplified from DNA of tumor and normal tissue. A panel of additional five MSI-markers is used for MSI classification of borderline cases. These numerous markers that require analysis of matched normal DNA of the same patient make MSI analysis a laborious and costly testing procedure that is not applicable for high throughput screening.
Therefore, a simplified testing strategy is required for high throughput testing. To reduce the workload of MSI testing, several techniques have been suggested in previous publications. Immunohistochemistry with monoclonal antibodies specific for MLH1 and MSH2 is commonly accepted as a useful tool to identify HNPCC-related tumors (Marcus et al. 1999 “Immunohistochemistry for hMLH1 and hMSH2: a practical test for DNA mismatch repair-deficient tumors, Am J Surg Pathol. 1999 October; 23(10):1248-55”, Lindor et al. “Immunohistochemistry versus microsatellite instability testing in phenotyping colorectal tumors”, J Clin Oncol. 2002 Feb. 15; 20(4):897-9; Umar et al. “Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability”, J Natl Cancer Inst. 2004 Feb. 18; 96 (4):261-8) and sensitivity can further be enhanced by the inclusion of additional antibodies recognizing MSH6 and PMS2. Compared to PCR-based MSI testing, IHC has some advantages, mainly the lower costs that were estimated to be less than one-third when compared to standard MSI analysis (Debniak et al. “Value of pedigree/clinical data, immunohistochemistry and microsatellite instability analyses in reducing the cost of determining hMLH1 and hMSH2 gene mutations in patients with colorectal cancer”, Eur J. Cancer. 2000 January; 36(1):49-54).
However, there are several limitations of IHC as a screening method when used alone. Some cases of MSI-H tumors are missed (Lindor et al. Immunohistochemistry versus microsatellite instability testing in phenotyping colorectal tumors”, J Clin Oncol. 2002 Feb. 15; 20(4):897-9), and false negative results have been reported due to intratumor heterogeneity, so staining of at least two independent samples for each carcinoma was recommended (Chapusot et al. “Microsatellite instability and intratumoural heterogeneity in 100 right-sided sporadic colon carcinomas”, Br J Cancer 2002 Aug. 12; 87(4):400-4). Furthermore, staining artifacts may result from formalin fixation procedure, especially when large tissue blocks are used (reviewed by Werner et al. 2000). Hence, the use of PCR-based MSI detection methods is indispensable for correct MSI classification at present. To minimize costs of PCR-based MSI testing, the use of BAT26 alone has been suggested in several studies (Zhou et al. “Determination of the replication error phenotype in human tumors without the requirement for matching normal DNA by analysis of mononucleotide repeat microsatellites”, Genes Chromosomes Cancer 1998 February; 21(2):101-7; Cravo et al. “BAT-26 identifies sporadic colorectal cancers with mutator phenotype: a correlative study with clinico-pathological features and mutations in mismatch repair genes”, J. Pathol. 1999 July; 188(3):252-7; Stone et al. “Optimising methods for determining RER status in colorectal cancers”, Cancer Lett. 2000 Feb. 28; 149(1-2):15-20), even without the need for matching normal tissue (Hoang et al. “BAT-26, an indicator of the replication error phenotype in colorectal cancers and cell lines”, Cancer Res. 1997 Jan. 15; 57(2):300-3). Although this approach may be sufficient for the majority of MSI-H cases, it does not equal the sensitivity of the ICG-HNPCC standard panel since there are false negative results. Additionally, depending on the ethnic origin of the tested individuals, shortened BAT26 alleles which have been reported in up to 5.3% (most frequent in Afro-American people, Pyaft et al. “Polymorphic variation at the BAT-25 and BAT-26 loci in individuals of African origin. Implications for microsatellite instability testing”, Am J. Pathol. 1999 August; 155(2):349-53) lead to false positive classification when corresponding normal tissue is not available (Perucho “Correspondence re: C. R. Boland et al., A National Cancer Institute workshop on microsatellite instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res., 58: 5248-5257, 1998.”, Cancer Res. 1999 Jan. 1; 59(1):249-56). Similarly, for BAT25 alleles aberrant from the general “wild type” have been detected in 0.6% to 6.8% of cases (Ichikawa et al. “DNA variants of BAT-25 in Japanese, a locus frequently used for analysis of microsatellite instability”, Jpn J Clin Oncol. 2001 July; 31 (7):346-8); Pyatt et al. “Polymorphic variation at the BAT-25 and BAT-26 loci in individuals of African origin. Implications for microsatellite instability testing”, Am J Pathol. 1999 August; 155(2):349-53). Therefore, Suraweera et al. (“Evaluation of tumor microsatellite instability using five quasimonomorphic mononucleotide repeats and pentaplex PCR”, Gastroenterology. 2002 December; 123(6):1804-11) recommended a pentaplex PCR system using BAT25, BAT26, and three additional mononucleotide markers that allowed reliable microsatellite typing in the majority of gastrointestinal tumors and cell lines that were tested. However, MSI status of a considerable number of tumors was pre-typed by only dinucleotide markers or BAT25/BAT26 alone, thus hampering the evaluation of the diagnostic sensitivity and specificity of the pentaplex system. Sutter et al. (Molecular screening of potential HNPCC patients using a multiplex microsatellite PCR system”, Mol Cell Probes. 1999 April; 13(2):157-65) recommended a combination of five markers in a multiplex system that reached 100% sensitivity and specificity, but only when used in combination with corresponding normal tissue.
The currently recommended procedure using the standard ICG-HNPCC marker panel for this purpose is costly and time-consuming. It is therefore desirable to establish a new microsatellite testing procedure. This procedure could e.g. include a novel marker highly indicative for MSI that could simplify the current protocols for MSI evaluation.
The compounds and methods disclosed according to the present invention provide for improvement of the microsatellite testing procedure. The procedure disclosed herein is prone to simplify MSI analysis in colorectal cancer without reducing the diagnostic sensitivity or specificity. The inventors found that the 3′-UTR T25 mononucleotide repeat of the CASP2 gene (in the following referred to as CAT25) may be used for an efficient and sensitive determination of the microsatellite status in specimens. In certain embodiments the disclosed marker may also be combined with the established microsatellite markers BAT25 and BAT26 in one multiplex amplification reaction.